Protease Inhibition
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An evolving field of applications of drug-related peptidomics is the analysis of protease inhibitors and their impact on the peptidome. To evaluate a protease as a potential drug target, it is beneficial to identify its substrates and products in vivo as this may impact the drug development process since specificity of inhibitors, and thus limiting side effects, can be predicted more accurately.
To learn more please download the presentation summarizing the utility of Peptidomics for protease inhibitor research.

The principle of protease inhibition plays an important role in drug discovery and modern medicine. The human genome contains more than 550 different proteases and peptidases, and also a considerable number of protease inhibitors. Since peptides are generated from larger precursors by the irreversible hydrolysis of a peptide bond, the peptidome reflects a natural library of substrates and products. Sequence analysis reveals the specific cleavage site present in the peptide or its precursor, and consequently this peptide is suspected to be a substrate or product of the inhibited enzyme. Consequently peptidomics is an excellent tool to evaluate a protease inhibitor in vivo by identifying its substrates and products and thus supporting the drug development process.
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The scheme depicts the effect of protease inhibitors on downstream products of the respective inhibited protease. On the other hand substrates of the protease are accumulated and may be processed by alternative pathways.

Influence of Protease Inhibition on Plasma Peptide Pattern
This example demonstrates the use of Peptidomics® as a suitable tool for the characterization of proteases and their inhibitors in vivo. We performed a study to evaluate the effect of protease inhibition on blood peptides in a rodent model. Rats were treated with a protease inhibitor or vehicle and blood samples were analyzed by Peptidomics®.
The treatment with the protease inhibitor resulted in a dose dependent decrease of protease activity.
More than 3,000 signals were shown in a mass range < 15 kDa. Correlation analysis with residual enzymatic activity showed that approx. 55 peptides were decreased or increased in abundance.
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The Figure shows a mean Peptide Display of analyzed plasma samples (n=27) of rats treated with a protease inhibitor (PI) or vehicle/saline. The inlet shows a Differential Peptide Display between plasma samples from treated rats (red) and control samples. The arrows mark two regulated peptides (red: increased in samples from rats treated with the PI, blue: increased in samples from rats treated with vehicle/saline).
The sequence analysis revealed both peptides which are marked in Figure 1

  • derived from the same precursor protein
  • the peptide marked with a blue arrow corresponds to a truncated form of the peptide marked with an red arrow
  • the cleavage site corresponds to the known protease specificity

Due to the truncation of the peptide a shift in the molecular mass hydrophobicity is visible in the Differential Peptide Display. To evaluate the abundance of both peptides all mass spectra of the corresponding region (molecular mass and hydrophobicity) are compiled:
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The figure shows the intensity of regulated peptides (squares) in 27 individual samples. The color of the squares refers to Figure 1. The indicated mass difference of 200 Dalton corresponds to truncation as determined by MS/Ms sequencing.
This example demonstrates the suitability of Peptidomics® to profile drug action of protease inhibitors in vivo providing a method to identify drug targets and potential adverse side effects.